Evaluation de régulateurs positifs de la croissance musculaire chez un modèle dystrophique murin

En 1997, le caractère culard, un phénotype hypermusclé chez le bovin, est attribué à des mutations dans le gène de la myostatine (MSTN). Depuis, il a été confirmé qu une baisse de l activité de la MSTN conduisait à une augmentation de la masse musculaire chez d autres espèces, y compris chez l Homme. L identification de ce facteur et des conséquences de son invalidation sur le développement musculaire ouvre de nombreuses perspectives en médecine humaine comme, par exemple, chez des personnes ayant eu une fonte musculaire importante suite à une immobilisation prolongée ou en conséquence du vieillissement ou d une maladie chronique. L objectif majeur de ce projet de recherche a consisté à évaluer de nouvelles stratégies permettant d augmenter la masse musculaire chez la souris. Pour ce faire, nous nous sommes intéressés à une métalloprotéine de la matrice extracellulaire (MEC), la décorine (DCN), dont l interaction avec la MSTN a été caractérisée comme étant zinc dépendante. Suite à l injection de ce Small Leucine Rich Proteoglycan (SLRP) chez la souris dystrophique mdx et Gamma-sarcoglycan-/-, nous avons constaté une augmentation de la masse musculaire consécutive à un phénomène d hypertrophie associé ou non à de l hyperplasie. Des études de dose/cinétique ont montré que l effet positif de la décorine sur la croissance musculaire était maximal 21 jours après administration. Nous avons également découvert qu un fragment peptidique de 41 acides aminés du domaine N-terminal de la protéine DCN murine conservait une activité anti-myostatine et induisait une hypertrophie musculaire chez la souris dystrophique. Ce domaine, site de l interaction directe entre la MSTN et la DCN, présente un motif CX3CXCX6C, caractéristique des SLRPs de classe I, dont le cluster de cystéines et son interaction avec le zinc ont été décrits comme indispensables à l activité anti-MSTN de la DCN. Différentes études concernant les mécanismes induits lors de la séquestration de la MSTN par la DCN dans la MEC ont également été conduites afin d expliquer les phénomènes observés chez la souris. Enfin, nous avons étudié le potentiel de la DCN pour favoriser la greffe de cellules myogéniques et développé différentes approches de thérapie génique.In 1997, the double-muscling phenotype, a marked hypermuscularity in cattle, was related to mutations in the myostatin (MSTN) gene. Since, it was confirmed that a decrease of the myostatin s activity drives an increase of the muscular mass in others species, including Human. The identification of this factor and the consequences of its invalidation on the muscular development open many perspectives in human medicine, as, for example, for people whom have an important muscular loss fallow up an extended immobilization or in consequence of old age or a chronic disease. The main purpose of this research project was to evaluate some new strategies permitting the increase of the muscular mass in mice. To achieve that, we investigated in detail the decorin (DCN), a metalloprotein of the extracellular matrix (ECM), interacting with MSTN in a zinc-dependent manner. After intramuscular injection of this Small Leucine Rich Proteoglycan (SLRP) in mdx and Gamma-Sarcoglycan-/- dystrophic mice, we observed a significant increase of the muscle mass conducted by hypertrophy associated or not with hyperplasia. Dose and cinetic studies showed that the positive effect of the decorin on muscular growth was maximal 21 days after administration. Furthermore, we showed that a peptide encompassing the 31-71 sequence retains full myostatin binding capacity and intramuscular injection of this peptide induces muscle hypertrophy in dystrophic mice. This direct interaction site between MSTN and DCN contains a conserved CX3CXCX6C pattern of class I SLRPs, whose cluster of cysteins and its interaction with zinc were shown to be crucial in the anti-MSTN activity of DCN. Various studies of the mechanism resulting of the sequestration of MSTN by DCN in ECM were conducted in order to explain the phenomenom observed in mice. Al last, we have studied the potential of DCN in the cellular transplantation and developped different anti-myostatin strategies of genetic therapy.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Hybrid Cell-Penetrating Foldamer with Superior Intracellular Delivery Properties and Serum Stability

International audienceSequence specific molecules with high folding ability (i.e. foldamers) can be used to precisely control the distribution and projection of side chains in space and have recently been introduced as tailored systems for delivering nucleic acids into cells. Designed oligourea sequences with an amphipathic distribution of Arg-and His-type residues were shown to form tight complexes with plasmid DNA, and to effectively promote the release of DNA from the endosomes. Herein, we report the synthesis of new cell-penetrating foldamer sequences in which the foldamer segment is conjugated via a reducible disulfide bond to a ligand that binds cell-surface expressed nucleoproteins with the idea that this system could facilitate both assemblies with nucleic acids and cell entry. This new system was evaluated for delivery of DNA in several cell lines and was found to compare favorably with all comparators tested (DOTAP and b-PEI as well as a number of known cell penetrating peptides) in various cell lines and particularly in culture medium containing up to 50% of serum. These results suggest that this dual molecular platform which is long lasting and non-cytotoxic could be of practical use for in vivo applications

Polydiacetylenic nanofibers as new siRNA vehicles for in vitro and in vivo delivery

Polydiacetylenic nanofibers (PDA-Nfs) obtained by photopolymerization of surfactant 1 were optimized for intracellular delivery of small interfering RNAs (siRNAs). PDA-Nfs/siRNA complexes efficiently silenced the oncogene Lim-1 in the renal cancer cells 786-O in vitro. Intraperitoneal injection of PDA-Nfs/siLim1 downregulated Lim-1 in subcutaneous tumor xenografts obtained with 786-O cells in nude mice. Thus, PDA-Nfs represent an innovative system for in vivo delivery of siRNAs

Polymers for Improving the In Vivo Transduction Efficiency of AAV2 Vectors

Background: Adeno-associated virus has attracted great attention as vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy infusion of very large amounts of vectors is required. This not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome these problems would be to find strategies able to increase the in vivo efficiency. Methodology: Here, we investigated whether polymers can act as adjuvants to increase the in vivo efficiency of AAV2. Our strategy consisted in the pre-injection of polymers before intravenous administration of mice with AAV2 encoding a murine secreted alkaline phosphatase (mSeAP). The transgene expression, vector biodistribution and tissue transduction were studied by quantification of the mSeAP protein and real time PCR. The injection of polyinosinic acid and polylysine resulted in an increase of plasmatic mSeAP of 2- and 12-fold, respectively. Interestingly, polyinosinic acid pre-injection significantly reduced the neutralizing antibody titer raised against AAV2. Conclusions: Our results show that the pre-injection of polymers can improve the overall transduction efficiency of systemically administered AAV2 and reduce the humoral response against the capsid proteins

Evaluation et optimisation de stratégies de correction génique

Une alternative attrayante à l approche traditionnelle de la thérapie génique par addition de gène, ou thérapie par les gènes, consiste à corriger in situ le gène muté. Cette approche par thérapie du gène, ou correction génique, offre potentiellement de nombreux avantages comparée à la thérapie génique traditionnelle. Malheureusement, bien que nombreux agents correcteurs soient disponibles aujourd hui, tels que des oligodéoxynucléotides simple-brins, des grands fragments d ADN simple- et double-brins ainsi que des vecteurs rAAV, peu d informations sont disponibles quant aux efficacités relatives de ces différentes approches sur un même système modèle. Nos travaux ont consisté tout d abord à évaluer les efficacités de correction génique de ces approches. Puis, nous avons montré que le prétraitement des cellules par des agents causant des dommages à l ADN permet d augmenter l efficacité de correction génique de ces approches.Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. Yet, an attractive alternative would be to correct the mutated gene that already exists in the affected individual. While many gene correction agents were described for their ability to correct mutations, no direct comparison of the repair efficiency has ever been made. Here, we present a side by side quantitative comparison of the correction efficiency of different forms of donor nucleic acids, including synthetic DNA oligodeoxynucleotides, large DNA fragments and sequences carried by a recombinant adeno-associated virus. Also, we show that a pretreatment of cells with doxorubicin (a topoisomerase II inhibitor) or with phleomycin (a glycopeptide antibiotic), both substances known to induce DNA damages, can increase the correction efficiency of the different repair tools.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Approches de thérapies géniques pour des maladies neuromusculaires

La thérapie génique de myopathies telles que la dystrophie musculaire de Duchenne nécessite une approche systémique afin de traiter l ensemble de la musculature. Le vecteur AAV est actuellement le plus efficace pour transduire le muscle. Nous montrons que la biodistribution du vecteur AAV administré par voie veineuse peut être modifiée en utilisant diverses stratégies adjuvantes chez la souris saine. La pré-injection de polymères permet ainsi d améliorer la transduction des muscles par le vecteur AAV, ou encore de baisser la réponse immune neutralisante induite par l injection intraveineuse du vecteur. Nous abordons également l impact de facteurs modulateurs exogènes ou endogènes tels que la procédure d administration ou certains facteurs sanguins sur la transduction systémique de l AAV. Dans une seconde approche, nous avons évalué le transfert de gènes dans le muscle dystrophique afin de sécréter dans la circulation sanguine une protéine transgénique fusionnant le récepteur soluble I du TNF-a avec le fragment constant d une immunoglobuline (TNFR-Is/mIgG1). La comparaison des cinétiques de sécrétion obtenu après le transfert de gène dans le muscle de souris saines ou de souris dystrophiques mdx indique que le contexte inflammatoire du muscle dystrophique favorise une réponse immune contre le transgène. Nous montrons que l expression et la sécrétion d un variant murin peu immunogène du TNFR-Is/mIgG1 améliore la fonction musculaire de la souris mdx sans toutefois conférer un avantage sélectif aux fibres musculaires dystrophiques qui continuent leur cycle de nécrose et de régénération.Gene therapy of myopathies such as Duchenne muscular dystrophy requires a systemic approach in order to treat the whole musculature. The AAV vector is currently the most efficient delivery system for muscle transduction. We show that the biodistribution of AAV administered intravenously can be modified using different adjuvant strategies in healthy mice. In particular, the pre-injection of polymers enables an improvement of muscle transduction by AAV, and can also decrease the neutralizing immune response induced by the intravenous injection of this vector. We also explored in this work the impact of exogenous and endogenous modulating factors such as the administration procedure or some blood factors on the AAV transduction capacity. In a second approach, we evaluated gene transfer in dystrophic muscles in order to secrete in the blood circulation a transgenic protein associating the soluble TNF-a receptor I and the Fc fragment of an immunoglobulin (TNFR-Is/mIgG1). The comparison of the kinetic of secretion after muscle gene transfer in healthy and dystrophic mice indicates that the inflammatory context of dystrophic muscle increases the immune response against the transgene. We also show that while the expression and secretion of a low immunogenic murine variant of TNFR-Is/mIgG1 improves the mdx muscle function, it does not confer a selective advantage to muscle fibers which still undergo cycles of necrosis and regeneration.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Fasting Increases the In Vivo Gene Delivery of AAV Vectors

International audienceSuccessful gene therapy of many genetic diseases requires efficient delivery of the gene to several tissues of the organism. Adeno-associated virus (AAV) is, to date, the sole vehicle that allows to achieving this result but only at the condition of administering very large amounts of vectors. This, however, raises questions about the feasibility of the large-scale production and about the safety of the approach. One way to overcome both problems would be to develop strategies that increase the in vivo efficiency. Here, we investigated the effect of fasting on the transduction efficiency of AAV serotypes 2, 6, and 9. The transgene expression was followed for several weeks and vector biodistribution was determined by real-time polymerase chain reaction (PCR) . The results show that fasting increases the transduction efficiency of all three serotypes. Altogether, we present here a simple and clinically acceptable approach that may allow to reducing the vector dose

Histidine-rich designer peptides of the LAH4 family promote cell delivery of a multitude of cargo

International audienc

A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair

<p>Abstract</p> <p>Background</p> <p>Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome.</p> <p>Results</p> <p>In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin.</p> <p>Conclusion</p> <p>Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.</p